Paper - The development of the blastoderm of the chick in vitro (1912)

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McWhorter JE. and Whipple AO. The development of the blastoderm of the chick in vitro. (1912) Anat. Rec. 8(3): 121-

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This historic 1912 paper by McWhorter and Whipple describes early experiments of in vitro chicken development.



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The Development of the Blastoderm of the Chick in vitro

John E. Mcwhorter and Allen O. Whipple

Columbia UniverfiHy, New York City

Twelve Figures

  • Read before the American Association of Anatomists, December 27, 1911, at Princeton, N. J.
  • From the Department of Surgery, College of Physicians and Surgeons, Columbia University (The George Crocker Special Research Fund).
  • From the Department of Surgery, College of Physicians and Surgeons, Columbia University
  • Expense of illustrations borne by authors. Anat. Record, vol. 1, 1907.

Introduction

Since Harrison's publication in 1907" in which he described the development in vitro of the nerve fibers of the frog embryo, experimental work in growing tissues has been largely confined to the culture of bits of tissue removed either from the embryo or adult animal or from various tumors. The development in vitro of the chick embryo, so far as we are aware, has not been described.

The rhythmical and vigorous contractions, for over five days, of the heart removed from a forty-hour chick embryo and planted in plasma, suggested to us the study of the developing vascular system. A seventy-two hour embryo was planted in plasma. The heart continued to beat uninterruptedly for over nine hours. During this time the entire vascular system could be seen in action under the microscope. While observing this first specimen we were impressed with the possibilities which such a method offers for the study of various problems in embryology and pathology. We began accordingly to test the viability and developmental possibilities of younger embryos. In this work we employed, in a modified form, the apparatus and technique which we have been using during the past year in growing adult and embryonic tissues.

The Apparatus

The modified incubator is shown in the accompanying photograph (fig. 1). Its chief advantages are: First, observation of the specimen on a mechanical stage controlled from the outside, without alterations in temperature; Secondly, the opportunity offered to take low and high power photomicrographs of all or parts of the specimen with an accurately focused, condensed, and heat filtered light.

The incubator consists of two parts: a base below, above this the incubator proper containing the microscope. The base is a rectangular sheet-iron box similar to the usual type excepting for an air space which is obtained by means of a heavy iron plate placed 7 cm. below the under surface of the incubator. The purpose of this air space is to maintain a more equable temperature within the incubator.

The in(^ubator proper, which rests upon an iron rim on the base, consists of a wooden box thoroughly insulated on its outer surfaces with asbestos. To its lower surface, is attached a perforated iron plate; the perforations are continuous with similar ones in the asbestos and wooden floor. These warm air inlets are plugged with glass tubes containing glass wool for filtering the air. Through the upper surface projects the draw tube of the microscope and immediately behind this is a small aperture for the extension rod connecting with the fine adjustment; to the right is a small door giving access to the incubator. A window in front transmits light to the mirror of the microscope. Above and to the side of this window is an opening for the controlling device of the Bean's heat regulator. At the back are two small openings, one above the other, for cords attached to the lever of the iris diaphragm. On the right side are extension rods controlling the coarse adjustment of the microscope and the two controls of the mechanical stage. On the left side is a door of ample size to allow free access to the incubator. Back of this passes an extension rod to control the substage. One arm of a right angled thermometer passes through this side and is so situated that its bulb lies immediately below stage of microscope. The temperature of the iiicuhutor is niaiiitainod by moans of tho small Bunscii burner in the base. The Bean's heat regulator as a thermostat has proved quite satisfactory as the tem])eraturc within the incubator seldom shows a variation of more than one degree.

The device here used for photomicrography consists, as is shown in the photograph, of a camera attached by means of a thumb screw to a slotted plate. This plate is kept rigid by being screwed to a frame work of iron piping, which in turn is securely fastened to the floor and braced. When taking photographs the camera is lowered to the lower limit of the slotted plate and the brass tube on the lens board of camera is inserted into the sleeve of the collar surrounding the draw tube of the microscope. When not in use the camera is disconnected from the microscope and pushed to the upper limit of the plate and clamped.

The method used for illuminating the field for photography is similar to that generally used in photomicrography, the only difference being that in this w^ork the use of a heat filter is essential, for the concentrated heat rays from the small arc lamp, if not filtered, cause tissue death in a very few seconds.

The Technique

Fresh eggs are incubated at 37° to 39° C. Using aseptic precautions throughout, the blastoderm is removed from the egg by cutting wide of the area vitellina externa and lifting it out of the 3'olk w^ith sufficient adherent yolk to prevent injury. The blastoderm is transferred to Locke's solution, kept at 37° C. on a water bath. Yolk granules and vitelline membrane are removed by gently squirtmg the solution against the blastoderm with a medicine dropper. The blastoderm is then floated onto a cover glass with its dorsal or upper surface in contact with the cover glass; the excess of Locke's solution is removed with sterile absorbent cotton. A few drops of plasma are placed on the blastodsrm and when this has coagulated the cover glass is inverted over a hollow glass slide, containing a drop of water, and rimmed w^ith paraffine. The specimen is then incubated at 38° C.


Plasma is obtained from the adult fowl by the method described by Carrel and Burrows, that is, blood is drawn from the external jugular vein through a glass canula, coated with olive oil, into ice cold parafhne tubes. The blood is centrifuged and the plasma is then refrigerated. We have used plasma either pure or mixed in different proportions with Locke's solution. When used unadulterated the specimen is securely fixed to the cover glass. This obviates the sagging and blurring of the specimen. The disadvantage in its use is the mechanical resistance which the jelly-like film, with it« fibrin network, necessarily offers to the free expansion of the embryo in its growth. By means of special glass slides in which the blastoderm rests on a depressed disc in a mixture of Locke's solution and plasma or serum the hanging drop jnethod is avoided. We hope to get better results by this means, which we have only recently employed.

The chief factors in preventing prolonged growth in our series of blastoderms have been:

  1. Injury: from rough handling, drying or cooling.
  2. The limited supply of oxygen and nutriment. This difficulty we expect to overcome, in part at least, by improvements in the chamber.
  3. The prolonged or too frequently repeated exposure of the blastoderm to the strong light in making photomicrographs. The plasma in many such cases liquefied much more rapidly than usual.


Observations

Our attempts to grow embryos before the appearance of the head fold have been unsuccessful, largely because of the difficulty of removing the blastoderm from the egg at such an early stage. But from the 3-4 somite stage up to 17 - 18 somites we have been able to watch continuous development. Embryos removed after the beginning of the heart beat, the 10 - 12 somite stage, have lived a variable length of time, the longest thirty-one hours. Comparing our specimens that have grown, as determined by the increase in somites and development of the nervous and vascular systems with those removed from the egg at corresponding stages


ol iiu'uhal ion it is cn idciit lli;it dcN (•Idpinciit ///. ////'o is |)r;ict ic;illy the sam(> as /// oro.

By this method coiit iiiuous ohsciA'atioiis, hitlici'lo impossible, can h(> made in the (h'X'elopment of the |)rimar>' divisions of the l)rain, tlie o|)ti{' and the otic Ncsicles. the rehition of tlie fohls of the anmion to tlie splanclmopleufe and somatopleufe, the folding of the heart, with the eephalic pn)«>;ressi()n of the auricles, and the process of somitic division. It is in the field of angiogenesis, however, that we believe the study of the blastoderm in vitro offers special opportunities.

We wish to make a preliminary re]:)ort on observations made in the blastoderm before the estal)lishment of well formed ))lood vessels.

In the area pellucida, with the embryo at the 3-6 somite stage, spaces appear, first at the margin of area opaca, shortly afterward in area pellucida and margin of embryonic body wall. The best place to observe them is in the area pellucida. Here they a]ipear at first as isolated spaces, of various shapes and sizes, in an undifferentiated layer of mesenchymal cells beneath the ectoderm (fig. 7,1,2,5). These spaces are frequently bounded by a mere line, more or less refractile in character (fig. 7, 1 and 2). In others the lumen is lined with rounded or oval cells w^hich later become fusiform and flattened (fig. 8, 2 and 4)- In not all of these spaces is there a well defined limiting wall. Manj^ of them end blindly in undifferentiated mesenchyme (fig. 7, 1 and 5).

Under observation, these isolated spaces have been seen to change their shape, to expand, and to unite with similar spaces (figs. 9, 10). In some specimens this union of isolated spaces is ciuite a rapid one and, unless observed during a limited period the process cannot be seen. After the 10 somite stage we have been unable to detect isolated spaces. With the confluence of spaces channels are formed (fig. 11). At times they show a fairly well defined lining of flattened cells. Under the strain of increasing fluid content these channels, situated as they are in the soft ooze of mesenchyme, dilate and a bulging of their walls occurs in their weaker parts. ^ATiere the bays, or recesses, of adjacent channels meet a communication is established and a plexus is formed. This plexus formation is first noted at the level of the anlage of the omphalo-mesenteric veins (fig. 4, 2). With the establishment of the heart beat plexus formation progresses rapidly. A remarkable example of the effect of the hydro-dynamics of the pumped fluid in these channels was seen in No. 36 of our series. After the heart had been beating steadily for five hours the plasma covering the blastoderm liquefied and the embryo sagged, while the area opaca remained adherent to the cover glass. This caused a kinking of the left omphalo-mesenteric vein. Immediately many of the channels in the area pellucida tributary to the kinked vein became engorged and the process of bulging of the walls in their weaker parts, with the formation of new channels became strikingly apparent. In two instances the walls of parallel channels were pushed together. In one case, after the walls had been in contact a half hour a new opening formed and blood cells rushed from the vessel of greater blockage into the more rapid stream of the other vessel. The same specimen showed vessels which had collapsed, owing to the shifting of the blood stream to other channels. Within an hour the cells lining these channels had lost their endothelial character and reverted to the undifferentiated type of mesenchymal cell.

The presence of a fluid in these channels was well demonstrated in several of our specimens. For prior to the establishment of the circulation isolated cells or groups of cells were seen changing their position in the lumen of the channels. With the onset of the heart beat there began a to and fro motion of these cells. Finally, as the circulation became established in a portion of the plexus these cells, or groups of cells breaking off from cell masses, suddenly shot through the channels as if an obstruction was suddenly removed. In the plexus, in the neighborhood of the omphalo-mesenteric veins, the direction of travel of these first cells was always from the area opaca to the sinus venosus. In one of our series (figs. 5 and 6) we were able to observe the development of the plexus in this region, the appearance of the heart beat and of the first two folds of the heart, the early oscillating movements of })lo()d cells with their later streaming toward the sinus Ncuosiis, and liiially the |)assa<;(' of Mood cells from the afca opaca. 1 liroimli t he |)l('\iis. to t lie sinus \'(miosus, t lifoujili t li(dicaft , ilown the left doi'sal aorta, out of tlic Nitclliiic ai'tci-y. hack to the aiva oj)a('a.


Tli(> i)luMU)in('na of the d('\-cloi)iii{'nt of isolated spaces in the area pellucida; tlieir confluence, resulting in channels; the elaboration of a plexus from these channels; the effect of the hydrodynamics of fluid, i)umi)ed by the heart through these plexuses, in establishing well defined blood vessels have been observed by us in om- blastoderms. In some cases several of the stages have been watched in the same embryo. As yet we have been unable to follow all of the successive stages in the same specimen.


Fig. 1 Photomicrograph of modified incui);if or.


Fig. 2 Photomicrograph of embryo at 6-7 somite stage. Note incomplete closure of neural fold. This specimen lived eighteen hours. X 28.


Fig. 3 Photomicrograph of same (■iiil)r\() as in fig. 2 shows 1!) 1 1 somites. .\ot( dcvclopinciit of fore bruin. This specimen developed heart beat. X 2S.


Fig. 4 Photomicrograph of embryo showing amnio-cardiac vesicles. X 28.


Fig. 5 Photomicrograph of embryo whovving 12 - 13 somites. Heart and partial circulation developed under observation. X 28.


Fig. 6 Photomicrograph of same embryo 14 - 15 somites with further folding of heart. X 28.


Fig. 7. Photomicrograph of isolated spaces 1-2-5. These walls are refractile showiiis few cells. Note merging of space 5 in undifferentiated mesenchynial cells. X 500.


Fig. 8 Photomicrograph of two isolated spaces 1-1. Note various shapes of cells 2 - 3 - Ji- lining the spaces. X 500.


Fig. 9 Photomicrograph of isohited spaces 1-2-3, about to unite. X 500.


Fig. 10 Photomicrograph of same spaces 1- 2-3,&s shown in fig. 9 now united. X 500.


Fig. 12 Photomicrograph of channels 1 - 1 with evaginations £ - £ going on to plexus formation. X oflO.


Cite this page: Hill, M.A. (2021, April 12) Embryology Paper - The development of the blastoderm of the chick in vitro (1912). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Paper_-_The_development_of_the_blastoderm_of_the_chick_in_vitro_(1912)

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